IMMUNOAFFINITY PURIFICATION OF RECOMBINANT HEPATITIS-B SURFACE-ANTIGEN FROM YEAST USING A MONOCLONAL-ANTIBODY

A murine monoclonal antibody developed for the purification of recombi nant hepatitis B surface antigen was immobilized on a chromatographic support and used to adsorb and purify the recombinant antigen from yea st. The adsorption-elution behaviour was first investigated using mono clonal antibody-coated enzyme-linked immunosorbent assay plates and pe rforming adsorption, washing and elution procedures with different elu tion agents. It was found that 3 M KSCN and 8 M urea at neutral pH dis rupted antigen-antibody interactions in both systems. The procedure fo r washing the immunoaffinity column was optimized, using different sal ts and detergents. The best results were obtained by applying the star ting material in 1 M NaCl and washing with the same buffer. The use of 0.1% sodium deoxycholate in the washing buffer reduced about 20-fold lipopolysaccharide contamination in the eluates as compared with washi ng without detergent. The relationship between bed height and the adso rption capacity of the column was studied, and it was found that the d ynamic capacity decreased twice on reducing its length/diameter ratio tenfold. The recovery of antigen was not affected by increasing the fl ow-rate up to 25 cm/h but decreased at higher values. Using the optimu m conditions, the affinity column was able to purify the recombinant h epatitis B surface antigen to more than 90% purity and a 65% antigen r ecovery was obtained.